In molecular biology and related fields, it is often desirable to make a double stranded nucleic acid such as a PCR product available for interaction with an oligonucleotide, e.g. a capture probe. This can e.g. be done by separating the two strands of the nucleic acid by increasing the temperature or by increasing the pH until the double stranded nucleic acid denatures. After denaturation, the capture probe may be added and the conditions reversed so that annealing of the capture probe can occur. However, if both strands are still present in the sample, they may renature and obviously compete with the capture probe. This of course decreases the efficiency of the capture process.
One solution is to physically separate the two strands of the nucleic acid before capturing one of the strands with the capture probe. However, this requires additional manipulations, which takes time and which may also lead to loss of material.
Thus, in PCR it would be advantageous to prevent the DNA polymerase from replicating all the way to the 3′end of the template, hence leaving single stranded 5′overhangs (as described in U.S. Pat. No. 5,525,494 (Zeneca Limited) 11. June 1996).
A common way of blocking the DNA polymerase, thus creating a single stranded overhang—but without the benefits on PCR efficacy and subsequent capture obtained by the present invention—is to insert a carbon linker between a PCR primer and a nonsense oligonucleotide sequence. The disadvantage compared to the present invention is the potential of the nonsense oligonucleotide sequence to flip-back onto the target nucleotide sequence due to the non-rigid structure of the carbon linker—thus interfering with the PCR polymerase. The carbon linkers can also coil-up and bring the nonsense oligonucleotide sequence and the primer sequence within close proximity allowing the PCR polymerase to read-through the linker. Such interference of the linker decreases subsequently the inter-assay uniformity of the multiplex PCR assay, and such interferences are not seen in present invention due to the rigid structure of the employed polymerase blocker.
WO9421820 describe a PCR method which uses primers with a non-replicable region to generate PCR products with single stranded overhangs (tails). The inventors used two non-base analogs (1,3 propanediol and 1,4-anhydro-2-deoxy-D-ribitol) in the PCR primers to prevent the polymerase from replicating all of nucleobases of the oligonucleotide used as primer, hence leaving a single stranded overhang corresponding to the nucleobases located at the 5′side of the non base analogs.
However, WO9421820 and U.S. Pat. No. 5,525,494 does not disclose polymerase blockers that benefit the PCR reaction in terms of specificity and sensitivity or that facilitate subsequent capture of the PCR product.